16S Copy Number

What is the 16S copy number and why it matters?

The 16S rRNA gene (16S) has been widely used as a phylogenetic marker, particularly important for the taxonomic profiling of microbiome samples. Unlike other genes that code for proteins, the 16S-coding gene may be present in multiple copies in a single cell. Obviously, a bacterial strain must have at least one gene encoding 16S, but the copy number can go up to 15 (see the below chart). There is a positive correlation between the genome size and 16S copy number.

16S copy numbers of bacteria in EzBioCloud database (generated from only complete genomes)

When we analyze microbiome data using 16S amplicon sequences, all quantitative measures are a form of NGS read counts that are assigned to the known taxa. In this case, we actually count the number of a marker gene, typically 16S, present in a microbiome sample. However, what we eventually want to know is not the number of 16S reads but the number of corresponding cells, or CFU (colony forming units).

Let’s assume that we are analyzing a human fecal sample. After sequencing, we obtained 100 reads assigned to Bacteroides fragilis and also 100 to Prevotella copri. Both species are frequently found in the human gut. Should we say that two species are present in equal numbers? According to the EzBioCloud database which provides information about 16S copy number, B. fragilis has 6 copies whereas P. copri contains 4 copies [Learn more for B. fragilis and P. copri]. If we consider this, the corrected ratio between B. fragilis and P. copri should be 3:2, not 1:1. The necessity of 16S copy number correction or normalization has been raised by several studies (Kembel et al., 2012; Angly et al., 2014; Vandeputte, et al. 2017).

How to correct taxonomic profile data using 16S copy numbers

The relative taxonomic compositional data of a microbiome sample can be corrected by simple calculation once we know the 16S copy numbers of all species. A problem is that we do not know these values for all species. To obtain accurate data, one or more complete genome sequence is required. Incomplete genome assemblies derived from short NGS reads contain either no or an inaccurate number of 16S gene sequences. At present, there are 3467 species represented by complete genome sequences (As of Dec. 2017). 16S copy numbers of the remaining species, including uncultured phylotypes, should be interpolated using the existing data.

A couple of algorithms were proposed to predict the missing 16S copy numbers (Langille et al., 2013; Angly et al., 2014). In EzBioCloud 16S-based MTP app, the PICRUSt algorithm (Langille et al., 2013) is used to generate the 16S copy number database for all species/phylotypes in the EzBioCloud 16S database (the below figure).

Prediction of 16S copy numbers using PICRUSt algorithm

Implementation in EzBioCloud 16S-based MTP

EzBioCloud 16S-based MTP allows you to instantly and interactively apply 16S copy number correction to the comparative analysis of multiple samples, the calculation of beta-diversity, and to Biomarker Discovery (e.g., LefSe) as well. Our database of 16S copy number is more comprehensive than any other database as we utilize an up-to-date version of the genome database (8,631 quality-controlled genomes of 3,302 species; as of March 2018).